The selection of endogenous genes in human postmortem tissues

Precisely determining the postmortem interval (PMI), which is crucial to criminal and forensic cases, is a research in which quantitative RT-PCR (also known as qRT-PCR or real-time RT-PCR) has been used to analyse gene expression levels and data normalisation should be required to eliminate the differences among the samples. Therefore, it is quite necessary to find stable molecular biological markers in PMI determination research. In this study, we compared nine commonly used endogenous markers (containing ACTB, GAPDH, B2M, U6, 18S rRNA, hsa-mir-1, hsa-mir-9, hsa-mir-194-1 and hsa-mir-203) in the 109 human tissue samples obtained from autopsy at the aim of finding stable markers in human tissues with consideration of the impact of parameters (PMI and cause of death). After RNA was extracted from four tissues (heart, brain, kidney, skin), the Ct values of nine endogenous markers were obtained by qRT-PCR and assessed by geNorm software. The results showed that U6, GAPDH and 18S rRNA were the suitable markers in our set of samples in various corpse conditions, that B2M and ACTB were reliable internal controls in heart tissue only, and that microRNAs had such high M values that they should not be chosen for endogenous control genes.     

不同温度下大鼠脑组织RNA降解与早期PMI的相关性

目的探讨大鼠脑组织8种RNA指标,在不同温度下的表达水平与早期死亡时间(PMI)的相关性。方法将222只SD大鼠随机分为对照组(死后0 h)和4个实验组,实验组断颈处死后分别置于5℃、15℃、25℃和35℃的环境中,于死后1~24 h内9个时间点提取脑组织RNA,利用实时荧光定量PCR技术检测8种RNA指标(β-actin、GAPDH、RPS29、18S rRNA、5S rRNA、U6 snRNA、miRNA-9及mi RNA-125b)的表达水平,ge Norm软件选取合适内参,SPSS软件对内参标准化RNA指标进行回归分析,R软件构建推断PMI的数学模型,另选6只已知PMI的SD大鼠予以验证。结果 5S rRNA、miR-9和mi R-125b表达稳定,可作为内参指标。β-actin和GAPDH具有良好的时序性降解规律,在24 h内随PMI延长不断降解。R软件拟合得ΔCt值随PMI和温度变化的数学模型可用以推断PMI。运用β-actin和GAPDH验证模型的平均误差率分别为14.1%和22.2%。结论β-actin和GAPDH表达水平与PMI和环境温度相关性良好。本研究建立的数学模型可为温度变化条件下的早期PMI推断提供参考。     

Variation in Developmental Time for Geographically Distinct Populations of the Common Green Bottle Fly,Lucilia sericata (Meigen)*

Abstract: Time between death and discovery of remains, or postmortem interval (PMI), can be assessed using blow fly maggot age. Forensic entomologists rely on published, often nonlocal, species‐specific developmental tables to determine maggot age. In a series of common garden experiments, we investigated the developmental rate variation between populations of Lucilia sericata collected from Sacramento, CA, San Diego, CA, and Easton, MA at 16°C, 26°C, and 36°C. For the 16°C trial the time measurement started at egg hatch, while for the higher temperatures the experiment began at oviposition; the wandering stage signified the endpoint for all experiments. The distribution of developmental times differed significantly (ANOVA, p < 0.001) between the three populations within each temperature treatment. We discovered that regional variation of developmental times within a blow fly species exists. This study demonstrates the importance of assembling local population‐specific developmental tables when estimating larval age to determine PMI.     

Effects of Different Temperatures on the Development of Dermestes Frischii and Dermestes Undulatus (Coleoptera,Dermestidae): Comparison Between Species

Dermestidae could be useful in forensic investigations to assess the PMI as adults and larvae colonize dried remains. We reared two species of Dermestidae (Dermestes frischii and Dermestes undulatus) to understand the effects of different temperatures on the length of their whole life cycle and on their immature stages. Both species were reared at 23°C ± 0.5, RH 75% and at 26°C ± 0.5, 75% RH. Our result shows that the temperature is the main factor that influences the development of those species; in fact, increasing temperature leads to a shorter development cycle (59.8 ± 0.5 and 38.1 ± 0.2 for D. frischii; 50.6 ± 0.6 and 36.2 ± 0.2 for D. undulatus). Furthermore, we found that the number of the molts before the pupa decreases from 5–7 to 5–6 for D. frischii and from 4–6 to 4–5 for D. undulatus, respectively, at 23°C and 26°C.     

A Study on the Estimation of Postmortem Interval Based on Environmental Temperature and Concentrations of Substance in Vitreous Humor

A method to determine postmortem interval (PMI) based on environmental temperature and the concentrations of vitreous humor (VH) molecules were explored. Rabbit carcasses were placed in a chamber at 5, 15, 25, or 35°C, and 80–100 μL of VH was collected with the double‐eye alternating micro‐sampling method every 12 h. A Roche DPPI biochemical analyzer was used to measure the concentrations of six substances in VH samples. The interpolation function model and mixed‐effect model were employed for data fitting to establish equations for PMI estimation. The concentrations of K⁺, P, Mg²⁺, creatinine (CRE), and urea nitrogen (UN) exhibited an upward trend with increasing PMI in all temperature groups, while the concentration of Ca²⁺ showed a downward trend. Validation results using K⁺ and Mg²⁺ ions revealed that the mixed‐effect model provided a better estimation than the interpolation function model using the data from our experiment. However, both models were able to estimate PMI using temperature and VH molecule concentrations.     

小鼠死后脑RNA降解与死亡时间的相关性

目的 探讨小鼠死后不同时间和温度下脑组织β-actin mRNA、18S rRNA的降解规律与死亡时间的关系.方法 将24只健康成年C57BL/6小鼠随机分为2组,每组12只,断颈法处死后分别置于不同的环境温度中(4℃、37℃),恒定环境湿度为80%.分别于死后即刻和0.5、2、6、24、48h取小鼠脑组织进行总RNA...     

Estimating Time Since Death from Postmortem Human Gut Microbial Communities

Postmortem succession of human‐associated microbial communities (“human microbiome”) has been suggested as a possible method for estimating postmortem interval (PMI) for forensic analyses. Here we evaluate human gut bacterial populations to determine quantifiable, time‐dependent changes postmortem. Gut microflora were repeatedly sampled from the proximal large intestine of 12 deceased human individuals as they decayed under environmental conditions. Three intestinal bacterial genera were quantified by quantitative PCR (qPCR) using group‐specific primers targeting 16S rRNA genes. Bacteroides and Lactobacillus relative abundances declined exponentially with increasing PMI at rates of N_t = 0.977e^?0.0144t (r² = 0.537, p < 0.001) and N_t = 0.019e^?0.0087t (r² = 0.396, p < 0.001), respectively, where N_t is relative abundance at time (t) in cumulative degree hours. Bifidobacterium relative abundances did not change significantly: N_t = 0.003e^?0.002t (r² = 0.033, p = 0.284). Therefore, Bacteroides and Lactobacillus abundances could be used as quantitative indicators of PMI.     

Development of a PCR High‐Resolution Melt Assay for Artemisia absinthium (Wormwood) and a Triplex Assay with Two Additional “Unregulated Legal High” Species Datura stramonium (Jimson Weed) and Merremia tuberosa (Hawaiian Woodrose),

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an “unregulated legal high” in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be “thujone‐free.” However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) high‐resolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three “unregulated legal high” species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42 ± 0.20°C, 83.88 ± 0.22°C, and 87.77 ± 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed.     

大鼠死后脑组织RNA降解与死亡时间推断的研究

目的探讨SD大鼠死后脑组织中甘油醛-3-磷酸脱氢酶(GAPDH)mRNA、18s rRNA含量的比值与死亡时间(PMI)的关系。方法SD大鼠处死后分别置于15℃、20℃环境中,采用TRIzol试剂提取SD大鼠在死后即刻、1、2、3、5、7d时脑组织中的总RNA,采用RT-PCR方法与凝胶图像分析技术检测18s rRNA和GAPDH mRNA的含量,并将二者的比值(GAPDHmRNA/18s rRNA)与PMI进行统计学回归分析。结果SD大鼠死后脑组织中GAPDH mRNA在2d时降解不明显,但到3、5、7d时降解程度显著增加,且随着温度的升高,降解速度增加;而18S rRNA降解缓慢,直到7d仍无显著性降解变化,且降解速度几乎不受温度的影响。对GAPDHmRNA/18s rRNA值(Y)与PMI(X)进行回归相关分析:20℃时,Y=0.62-0.004X(R=-0.981,P<0.001);15℃时,Y=0.965-0.001X(R=-0.709,P<0.001)。结论SD大鼠死后脑组织中GAPDHmRNA和18s rRNA基因的相对稳定性即GAPDHmRNA/18s rRNA值与PMI存在明显相关性。     

不同死因大鼠心肌内多种RNA相对表达量变化与PMI的关系

目的观察不同死因下大鼠心肌内多种RNA相对表达量随死亡时间（PMI）的变化。方法建立断颈死、窒息死和失血性休克死大鼠模型,提取心肌中总RNA,利用实时荧光定量PCR技术检测甘油醛-3-磷酸脱氢酶（glyceraldehyde～3-phosphatedehydrogenase,GAPDH）,β-肌动蛋白（β-actin）、诱导型-氧化氮合酶（induciblenitricoxidesynthase。iNOS）、缺氧诱导因子-1（hypoxia-induciblefactor-1,HIF-1）、肿瘤坏死因子-d（tumornecrosisfactor-d,TNF-a）、白细胞介素-6（interleukin-6,IL-6）tuRNA以及U6微核RNAfU6sreallnuclearRNA。U6snRNA）的循环阈值,观察各指标相对表达量随PMI的变化情况。结果U6snRNA表达稳定,可作为内对照。在死亡早期,CAPDH、HIF-1、iNOS、TNF-d和IL-6的相对表达量在窒息组和失血性休克组比断颈组呈现不同程度的增加,而β-actin在3个组皆呈现相对表达量下降趋势。在死亡晚期,随着RNA的不断降解,相对表达量持续下降。结论死后各RNA相对表达量的特征性变化可为不同死因下的PMI推断提供-定的参考。     

Influence of Paraquat on Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) Infesting Minced‐beef Substrates in Kelantan,Malaysia

This study investigated the influence of paraquat, a prevalent poison used by suicides, on initial oviposition and development of Chrysomya megacephala (Fabricius) using minced‐beef substrates. Paraquat in lethal dose for human (40 mg/kg), two times the lethal dose (80 mg/kg) and five times the lethal dose (200 mg/kg) were mixed thoroughly with respective minced‐beef substrates (1 kg each) that were decomposed in a shaded habitat fully protected from rain. Results of four replications of the above experiment revealed that the presence of paraquat neither delayed initial oviposition nor prolonged the developmental stages of C. megacephala. Therefore, estimation of postmortem interval (PMI) based on empirical baseline data obtained using animal models devoid of any poisons would still be appropriate for estimating PMI in paraquat‐related deaths.     

外温变化下小鼠肝细胞18s rRNA降解与死亡时间关系研究

目的探讨在外界环境温度变化条件下,小鼠肝细胞18s rRNA降解与死亡时间的关系。方法小鼠断颈处死后置于10℃、15℃、20℃、25℃、和30℃温度下保存;从死后6h至72h,每6h分别提取肝细胞组织中总RNA。利用实时荧光定量PCR检测18s rRNA循环阈值(Ct值)的变化。应用插值函数进行分析拟合,建立死亡时间推断方程。结果各温度组Ct值随死亡时间延长均呈上升趋势,对所得数据进行插值拟合,得到一定温度变化区间内(10℃~30℃),Ct值变化与PMI关系的三元五次曲面方程:f(x,y)=-426.9+30.82x+44.48y-1.297x~2-1.837xy-1.388y~2+0.034 38x~3+0.038 17x~2y+0.038 67xy~2+0.028 77y3-0.000 612 9x~4-3.897e~(-7)x~3y-0.001 223x~2y~2+0.000256 6xy3-0.000 537 4y~4+3.606e~(-6)x~5-2.846e~(-6)x~4y+1.009e~(-5)x~3y~2-3.439e~(-6)x~2y3-2.556e~(-7)xy~4+2.664e~(-6)y~5(r2=0.999 4)。结论在外界温度变化条件下,小鼠肝细胞18s rRNA降解与死亡时间关系符合三元五次方程分布,利用插值函数拟合的方法可在外界环境温度变化条件下进行死亡时间推断。     

Temperature‐dependent Development of Parasarcophaga similis (Meade 1876) and its Significance in Estimating Postmortem Interval

Flesh flies are commonly found insects on decaying corpses that appears slightly later than blowflies, and their development patterns are significant indicators for minimum postmortem interval (PMI_min) estimation. In this study, the flesh fly Parasarcophaga similis (Meade 1876) was reared at nine constant temperatures ranging from 15°C to 35°C to examine indicators for estimating their age. We generated three development models, including isomorphen diagram, isomegalen diagram, and thermal summation model. Larval body length at different rearing temperatures was fit into an L = a + bT + cT² + dT³ equation with which the relationship between the larval body length (L) and the time after larviposition (T) was confirmed. The pupal stage was categorized into 13 substages according to intrapuparial morphological changes, and a detailed table was generated of the pupal developmental stages at five rearing temperatures, 15°C, 20°C, 25°C, 30°C, and 35°C. This study provides fundamental data in supporting P. similis as an indicator for PMI_min estimation.     

Response Assessment of Thermal Papers from Four Continents to Fingerprint Development by Heat

Latent fingerprint deposits on thermal paper sourced from the U.S., China, the U.K., and Australia have been visualized by heating. U.S. and Chinese sourced paper produced two distinct types of fingerprint development. In one type (type 1), the paper dye colors where the deposit is present (as previously reported) and in the other type (type 2) the ‘inverse’ of this gives paper coloring only in areas not coincident with the deposit. Both development types gave identifiable fingerprints, the majority fading within 24 h of heating. Fingerprint development from U.K. and Australian sourced paper was exclusively type 1 and resistant to fading. Temperatures for fingerprint visualization were higher for U.S. paper (64–71°C) and Chinese paper (75–95°C) than for U.K. and Australian sourced paper (43–50°C). Particularly for Chinese sourced paper, these temperatures were within a few degrees of the normal paper color temperature. A mechanism for type 2 fingerprint development is proposed.     

The Effect of Soft Tissue on Temperature Estimation from Burnt Bone Using Fourier Transform Infrared Spectroscopy

This study investigated the effect of soft tissue and different exposure times on the prediction of burning temperatures of bone when using Fourier transform infrared spectroscopy (FTIR). Ovis aries rib bones were burnt at different temperatures and for varying time intervals. Results of a linear regression analysis indicated that burn temperatures can be predicted with a standard error of ±70°C from defleshed bone spectra. Exposure time does not have a significant impact on prediction accuracy. The presence of soft tissue has a significant impact on heat‐induced changes of the bone matrix in low (<300°C) as well as high temperatures (>800°C), slowing down combustion in the former and accelerating it in the latter (p < 0.05). At medium temperatures, no significant difference was noted. These results provide forensic investigators a new perspective with which to interpret the results of crystallinity measures derived from burnt bone.     

Bacterial Degradation of Risperidone and Paliperidone in Decomposing Blood

The stability of two benzisoxazole antipsychotics was determined in vitro in decomposing porcine blood inoculated with bacteria, utilizing a high‐performance liquid chromatography with ultraviolet and fluorescence detection method for drug quantitation. Stability experiments for risperidone and paliperidone were conducted at 7, 20 and 37°C for 4 days using sterile and bacterially inoculated porcine blood. The drugs were stable in sterile blood at each temperature and in inoculated blood at 7°C, but degraded significantly in inoculated blood at 20 and 37°C. Complete loss occurred within 2 days when incubated at 37°C. The benzisoxazole‐cleaved degradation products for both drugs were identified as 2‐hydroxybenzoyl‐risperidone and 2‐hydroxybenzoyl‐paliperidone utilizing liquid chromatography quadrupole‐time‐of‐flight mass spectrometry and accurate mass measurements. The degradation products have been found in postmortem case studies, including one case where risperidone and paliperidone were not detected, indicating complete conversion can occur in situ.     

Generating a Vertebrae‐based Method to Discriminate between Korean and U.S. White Male Casualties from the Korean War,

Low recovery rate of intact crania in conflict‐related contexts necessitates the use of postcranial elements for ancestry estimation. This study aimed to develop a new method to discriminate between Korean and U.S. White casualties from the Korean War using vertebrae. Maximum body heights of C2 through L5 were measured from 75 Korean and 51 U.S. males to generate discriminant functions. U.S. vertebral heights were statistically greater than Koreans in 18 out of 23 vertebrae (C2, C4‐6, T3‐6, and T8‐L5). The functions with lumbar vertebrae tended to yield higher correct classification ratios (CCR) than those with cervical or thoracic vertebrae. Inclusion of the femur and tibia lengths in the vertebrae‐involved functions enhanced the CCR's. Discriminant functions using the femur and tibia length were also presented. The results of this study are expected to complement the current practices of the Defense POW/MIA Accounting Agency and the Ministry of National Defense Agency for KIA Recovery and Identification, which are primarily based on cranial and dental morphology.     

Effect of Heat on the Fluorescence Properties of Tooth‐Colored Restorative Materials and Their Forensic Implications

During antemortem and postmortem comparison of dental records of carbonized victims, restorations may be part of such records. The objective of this study was to evaluate the effect of heat on the fluorescence behavior of contemporary tooth‐colored restorative materials and natural tooth structure when subjected to range of temperatures, using illumination with 405 nm wavelength light. A total of 132 human extracted teeth restored with tooth‐colored restorative materials were exposed to heat (200, 500, 900, 1200°C) in an oven for 30 min. Samples were imaged before and after heat treatment. All tooth‐colored restorative materials underwent changes in color and in fluorescence properties, at each of the temperatures used. Resin‐based restorative materials still fluoresced at 200°C, and at 500°C underwent major color changes due to volatilization of resin. Materials containing inorganic fluorophores still fluoresced at 900°C, while at 1200°C, none of the materials tested in this study showed any fluorescence.     

实时RT-PCR检测大鼠死后管家基因mRNA的时序性降解

目的研究实时荧光定量RT—PCR方法检测死亡大鼠管家基因mRNA时序性降解的可行性,为死亡时间（Dostmortem interval,PMI）推断寻找新的研究手段。方法应用SYBR Green Ⅰ实时荧光定量RT—PCR技术．检测死后不同时间大鼠脑和脾中管家基因GAPDHmRNA及β—actinmRNA的水平,结果用循环阈值（简称Ct值）表示,分析死后经过时间与Ct值的线性关系,并建立死亡时间推断回归方程。结果GAPDH mRNA和β—actinmRNA的Ct值均与PMI之间存在显著的相关性。结论SYBR GreenⅠ实时荧光定量RT—PCR在定量分析mRNA降解的研究中是一个较理想的技术手段。选用管家基因作为PMI推断的研究对象,可在法医检案中消除其他基因因为个体差异带来的误差,更具实用性。Ct值作为动态监测机体死后不同时间点的客观指标．与死后不同时间点的线性关系良好,推断死后经过时间尤其是晚期死亡时间较为理想。     

Bioelectrical Impedance as a Technique for Estimating Postmortem Interval,,

Estimation of postmortem interval (PMI) is a critical component of death investigation. A cadaver can be hypothesized to be a resistor–capacitor (RC) circuit the impedance (Z) of which changes in a quantifiable manner as the cadaver decomposes. This hypothesis was tested using bioelectrical impedance analysis (BIA) equipment to apply a current with a fixed amplitude at a single frequency to four cadavers over time and measuring two components of Z, resistance (R) and reactance (X_c). Quadratic regression analysis between Z and accumulated degree days (ADD) showed a statistically significant parabolic relationship. The parabolic relationship poses an initial challenge to the use of the method, and additional research is needed to address this issue. However, the results of the reported research support the hypothesis that Z measured using BIA has a relationship to PMI.